Nucleic acids encoding polypeptides having haloperoxidase activity

ABSTRACT

The present invention relates to isolated nucleic acid sequences encoding polypeptides having haloperoxidase activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/832,614, filed on Apr. 11, 2001 and claims priority under 35U.S.C. 119 from Danish application PA 2000 00626, filed Apr. 14, 2000,and the benefit of U.S. provisional application Ser. No. 60/202,249,filed May 5, 2000, the contents of which are fully incorporated hereinby reference.

STATEMENT REGARDING FEDERALLY-FUNDED RESEARCH OR DEVELOPMENT

[0002] The government has certain rights to this invention pursuant toGrant No. N65236-99-1-5418, awarded by SPAWAR.

FIELD OF THE INVENTION

[0003] The present invention relates to isolated nucleic acid sequencesencoding polypeptides having haloperoxidase activity. The invention alsorelates to nucleic acid constructs, vectors, and host cells comprisingthe nucleic acid sequences.

BACKGROUND

[0004] Haloperoxidases are widespread in nature being produced bymammals, plants, algae, lichen, bacteria, and fungi. Haloperoxidases areprobably the enzymes responsible for the formation of naturallyoccurring halogenated compounds. There are three types ofhaloperoxidases, classified according to their specificity for halideions: Chloroperoxidases (E.C. 1.11.1.10) which catalyze thechlorination, bromination and iodination of compounds; bromoperoxidaseswhich show specificity for bromide and iodide ions; and iodoperoxidases(E.C. 1.11.1.8) which solely catalyze the oxidation of iodide ions.

[0005] The first discovered haloperoxidases were determined to containheme as a prosthetic group or co-factor. However, more recently, it hasbecome apparent that there are numerous non-heme haloperoxidases aswell. Bacterial haloperoxidases have been found with no prostheticgroup. In addition, a number of other non-heme haloperoxidases have beenshown to possess a vanadium prosthetic group. Haloperoxidases containinga vanadium prosthetic group are known to include at least two types offungal chloroperoxidases from Curvularia inaequalis (van Schijndel etal., 1993, Biochimica Biophysica Acta 1161:249-256; Simons et al., 1995,European Journal of Biochemistry 229: 566-574; WO 95/27046) andCurvularia verruculosa (WO 97/04102).

[0006] Haloperoxidases, like other oxidoreductases, are of currentinterest because of their broad range of potential industrial uses.

[0007] It is an object of the present invention to provide improvedpolypeptides having haloperoxidase activity and nucleic acid encodingthe polypeptides.

SUMMARY OF THE INVENTION

[0008] The present invention relates to isolated nucleic acid sequencesencoding polypeptides having haloperoxidase activity selected from thegroup consisting of:

[0009] (a) a polypeptide having an amino acid sequence which has atleast 80% homology with the amino acid sequence of SEQ ID NO:2;

[0010] (b) a polypeptide encoded by a nucleic acid sequence whichhybridizes under medium stringency conditions with (i) the nucleotidesequence of SEQ ID NO:1, (ii) a subsequence of (i) of at least 100nucleotides, or (iii) a complementary strand of (i) or (ii);

[0011] (c) a variant of the polypeptide having an amino acid sequence ofSEQ ID NO:2 comprising a substitution, deletion, and/or insertion of oneor more amino acids;

[0012] (d) an allelic variant of (a) or (b);

[0013] (e) a fragment of (a), (b), or (d) that has haloperoxidaseactivity; and

[0014] (f) a polypeptide having more than 50% residual activity after 15minutes incubation at 70° C. and pH 7.

[0015] The present invention also relates to nucleic acid constructs,vectors, and host cells comprising the nucleic acid sequences.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having HaloperoxidaseActivity

[0016] The term “haloperoxidase activity” as defined herein catalyzesthe oxidation of a halide ion (X=Cl—, Br—, or I—) in the presence ofhydrogen peroxide (H₂O₂) to the corresponding hypohalous acid (HOX):

H₂O₂+X—+H+→H₂O+HOX

[0017] For purposes of the present invention, haloperoxidase activity isdetermined according to the procedure described in “Haloperoxidaseassays” in the Examples.

[0018] In a first embodiment, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofhomology to the amino acid sequence of SEQ ID NO:2 of at least about80%, preferably at least about 90%, more preferably at least about 95%,and most preferably at least about 97%, which have haloperoxidaseactivity (hereinafter “homologous polypeptides”). In a preferredembodiment, the homologous polypeptides have an amino acid sequencewhich differs by five amino acids, preferably by four amino acids, morepreferably by three amino acids, even more preferably by two aminoacids, and most preferably by one amino acid from the amino acidsequence of SEQ ID NO:2. For purposes of the present invention, thedegree of homology between two amino acid sequences is determined byusing GAP version 8 from the GCG package (Genetics Computer Group, 575Science Drive, Madison, Wis. 53711, USA) with standard penalties forproteins: GAP weight 3.00, length weight 0.100, Matrix described inGribskov and Burgess, Nucl. Acids Res. 14(16); 6745-6763 (1986).

[0019] Preferably, the polypeptides of the present invention comprisethe amino acid sequence of SEQ ID NO:2 or an allelic variant thereof; ora fragment thereof that has haloperoxidase activity. In a more preferredembodiment, the polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO:2. In another preferred embodiment, thepolypeptide of the present invention consists of the amino acid sequenceof SEQ ID NO:2 or an allelic variant thereof; or a fragment thereof thathas haloperoxidase activity. In another preferred embodiment, thepolypeptide of the present invention consists of the amino acid sequenceof SEQ ID NO:2.

[0020] A fragment of SEQ ID NO:2 is a polypeptide having one or moreamino acids deleted from the amino and/or carboxyl terminus of thisamino acid sequence.

[0021] An allelic variant denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

[0022] In a second embodiment, the present invention relates to isolatedpolypeptides having haloperoxidase activity which are encoded by nucleicacid sequences which hybridize under medium stringency conditions,preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with a nucleic acid probe which hybridizes under the sameconditions with (i) the nucleotide sequence of SEQ ID NO:1, (ii) asubsequence of (i), or (iii) a complementary strand of (i) or (ii) (J.Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Thesubsequence of SEQ ID NO:1 may be at least 100 nucleotides or preferablyat least 200 nucleotides. Moreover, the subsequence may encode apolypeptide fragment, which has haloperoxidase activity. Thepolypeptides may also be allelic variants or fragments of thepolypeptides that have haloperoxidase activity.

[0023] The nucleic acid sequence of SEQ ID NO:1 or a subsequencethereof, as well as the amino acid sequence of SEQ ID NO:2 or a fragmentthereof, may be used to design a nucleic acid probe to identify andclone DNA encoding polypeptides having haloperoxidase activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 15, preferably at least25, and more preferably at least 35 nucleotides in length. Longer probescan also be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

[0024] Thus, a genomic DNA or cDNA library prepared from such otherorganisms may be screened for DNA, which hybridizes with the probesdescribed above and which encodes a polypeptide having haloperoxidaseactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO:1 or a subsequence thereof, the carriermaterial is used in a Southern blot. For purposes of the presentinvention, hybridization indicates that the nucleic acid sequencehybridizes to a labeled nucleic acid probe corresponding to the nucleicacid sequence shown in SEQ ID NO:1, its complementary strand, or asubsequence thereof, under low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions are detected using X-ray film.

[0025] In a preferred embodiment, the nucleic acid probe is a nucleicacid sequence which encodes the polypeptide of SEQ ID NO:2, or asubsequence thereof. In another preferred embodiment, the nucleic acidprobe is SEQ ID NO:1. In another preferred embodiment, the nucleic acidprobe is the nucleic acid sequence contained in the pUC19 derivedplasmid contained in Escherichia coli DH10B, deposited as DSM 13442,wherein the nucleic acid sequence encodes a polypeptide havinghaloperoxidase activity.

[0026] For long probes of at least 100 nucleotides in length, low tovery high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for lowstringencies, 35% formamide for medium and medium-high stringencies, or50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

[0027] For long probes of at least 100 nucleotides in length, thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS preferably at least at 50° C. (low stringency), morepreferably at least at 55° C. (medium stringency), more preferably atleast at 60° C. (medium-high stringency), even more preferably at leastat 65° C. (high stringency), and most preferably at least at 70° C.(very high stringency).

[0028] For short probes which are about 15 nucleotides to about 70nucleotides in length, stringency conditions are defined asprehybridization, hybridization, and washing post-hybridization at about5° C. to about 10° C. below the calculated T_(m) using the calculationaccording to Bolton and McCarthy (1962, Proceedings of the NationalAcademy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6,6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodiumpyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mgof yeast RNA per ml following standard Southern blotting procedures.

[0029] For short probes which are about 15 nucleotides to about 70nucleotides in length, the carrier material is washed once in 6× SSCplus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6× SSCat 5° C. to 10° C. below the calculated T_(m).

[0030] In a third embodiment, the present invention relates to variantsof the polypeptide having an amino acid sequence of SEQ ID NO:2comprising a substitution, deletion, and/or insertion of one or moreamino acids.

[0031] The amino acid sequences of the variant polypeptides may differfrom the amino acid sequence of SEQ ID NO:2 by an insertion or deletionof one or more amino acid residues and/or the substitution of one ormore amino acid residues by different amino acid residues. Preferably,amino acid changes are of a minor nature, that is conservative aminoacid substitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

[0032] Examples of conservative substitutions are within the group ofbasic amino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions, which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

[0033] In a fourth embodiment, the present invention relates to isolatedpolypeptides having a residual activity of at least 50% residualactivity, preferably at least 60% residual activity after 15 minutesincubation at 70° C. and pH 7. In a preferred embodiment, thepolypeptides of the invention retain at least 50% residual activity,preferably at least 80% residual activity after 15 minutes incubation at60° C. and pH 7.

[0034] In a preferred embodiment, the polypeptides of the inventioncontain a vanadium prosthetic group, and accordingly they are vanadiumhaloperoxidases. In another preferred embodiment, the polypeptides ofthe invention are chloroperoxidases.

[0035] In a fifth embodiment, the present invention relates to isolatedpolypeptides having immunochemical identity or partial immunochemicalidentity to the polypeptide having the amino acid sequence of SEQ IDNO:2. The immunochemical properties are determined by immunologicalcross-reaction identity tests by the well-known Ouchterlony doubleimmunodiffusion procedure. Specifically, an antiserum containingpolyclonal antibodies which are immunoreactive or bind to epitopes ofthe polypeptide having the amino acid sequence of SEQ ID NO:2 areprepared by immunizing rabbits (or other rodents) according to theprocedure described by Harboe and Ingild, In N. H. Axelsen, J. Krll, andB. Weeks, editors, A Manual of Quantitative Immunoelectrophoresis,Blackwell Scientific Publications, 1973, Chapter 23, or Johnstone andThorpe, Immunochemistry in Practice, Blackwell Scientific Publications,1982 (more specifically pages 27-31). A polypeptide havingimmunochemical identity is a polypeptide, which reacts with theantiserum in an identical fashion such as total fusion of precipitates,identical precipitate morphology, and/or identical electrophoreticmobility using a specific immunochemical technique. A furtherexplanation of immunochemical identity is described by Axelsen, Bock,and Krll, In N. H. Axelsen, J. Krll, and B. Weeks, editors, A Manual ofQuantitative Immunoelectrophoresis, Blackwell Scientific Publications,1973, Chapter 10. A polypeptide having partial immunochemical identityis a polypeptide, which reacts with the antiserum in a partiallyidentical fashion such as partial fusion of precipitates, partiallyidentical precipitate morphology, and/or partially identicalelectrophoretic mobility using a specific immunochemical technique. Afurther explanation of partial immunochemical identity is described byBock and Axelsen, In N. H. Axelsen, J. Krll, and B. Weeks, editors, AManual of Quantitative Immunoelectrophoresis, Blackwell ScientificPublications, 1973, Chapter 11.

[0036] The antibody may also be a monoclonal antibody. Monoclonalantibodies may be prepared and used, e.g., according to the methods ofE. Harlow and D. Lane, editors, 1988, Antibodies, A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

[0037] The polypeptides of the present invention have at least 20%,preferably at least 40%, more preferably at least 60%, even morepreferably at least 80%, even more preferably at least 90%, and mostpreferably at least 100% of the haloperoxidase activity of thepolypeptide of SEQ ID NO:2.

[0038] A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by the nucleic acid sequence isproduced by the source or by a cell in which the nucleic acid sequencefrom the source has been inserted. In a preferred embodiment, thepolypeptide is secreted extracellularly.

[0039] A polypeptide of the present invention may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide; or a Streptomyces polypeptide, e.g., a Streptomyceslividans or Streptomyces murinus polypeptide; or a gram negativebacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

[0040] A polypeptide of the present invention may be a fungalpolypeptide, and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide; or more preferably a filamentous fungal polypeptide such asan Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide.

[0041] In a preferred embodiment, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis polypeptide.

[0042] In another preferred embodiment, the polypeptide is anAspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum, Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum,Trichoderma koningli, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide.

[0043] In a preferred embodiment, the polypeptide is a Geniculosporiumsp. polypeptide, more preferably a Geniculosporium sp. haloperoxidase,and most preferably a Geniculosporium sp. haloperoxidase encoded by thenucleic acid sequence contained in the plasmid contained in E. coliDH10B, deposited as DSM 13442, e.g., the polypeptide with the amino acidsequence of SEQ ID NO:2.

[0044] It will be understood that for the aforementioned species, theinvention encompasses both the perfect and imperfect states, and othertaxonomic equivalents, e.g., anamorphs, regardless of the species nameby which they are known. Those skilled in the art will readily recognizethe identity of appropriate equivalents.

[0045] Strains of these species are readily accessible to the public ina number of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammiung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

[0046] Furthermore, such polypeptides may be identified and obtainedfrom other sources including microorganisms isolated from nature (e.g.,soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms from natural habitats are wellknown in the art. The nucleic acid sequence may then be derived bysimilarly screening a genomic or cDNA library of another microorganism.Once a nucleic acid sequence encoding a polypeptide has been detectedwith the probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

[0047] As defined herein, an “isolated” polypeptide is a polypeptidewhich is essentially free of other non-haloperoxidase polypeptides,e.g., at least about 20% pure, preferably at least about 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by SDS-PAGE.

[0048] Polypeptides encoded by nucleic acid sequences of the presentinvention also include fused polypeptides or cleavable fusionpolypeptides in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide or fragment thereof. A fusedpolypeptide is produced by fusing a nucleic acid sequence (or a portionthereof) encoding another polypeptide to a nucleic acid sequence (or aportion thereof) of the present invention. Techniques for producingfusion polypeptides are known in the art, and include ligating thecoding sequences encoding the polypeptides so that they are in frame andthat expression of the fused polypeptide is under control of the samepromoter(s) and terminator.

Nucleic Acid Sequences

[0049] The present invention also relates to isolated nucleic acidsequences, which encode a polypeptide of the present invention. In apreferred embodiment, the nucleic acid sequence is set forth in SEQ IDNO:1. In another more preferred embodiment, the nucleic acid sequence isthe sequence contained in the pUC19 derived plasmid contained inEscherichia coli DH10B, deposited as DSM 13442. The present inventionalso encompasses nucleic acid sequences which encode a polypeptidehaving the amino acid sequence of SEQ ID NO:2, which differ from SEQ IDNO:1 by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO:1 which encodefragments of SEQ ID NO:2 that have haloperoxidase activity.

[0050] A subsequence of SEQ ID NO:1 is a nucleic acid sequenceencompassed by SEQ ID NO:1 except that one or more nucleotides from the5′ and/or 3′ end have been deleted.

[0051] The present invention also relates to mutant nucleic acidsequences comprising at least one mutation in the polypeptide codingsequence of SEQ ID NO:1, in which the mutant nucleic acid sequenceencodes a polypeptide which consists of the amino acid sequence of SEQID NO:2.

[0052] The techniques used to isolate or clone a nucleic acid sequenceencoding a polypeptide are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the nucleic acid sequences of the present invention from suchgenomic DNA can be effected, e.g., by using the well known polymerasechain reaction (PCR) or antibody screening of expression libraries todetect cloned DNA fragments with shared structural features. See, e.g.,Innis et al., 1990, PCR: A Guide to Methods and Application, AcademicPress, New York. Other nucleic acid amplification procedures such asligase chain reaction (LCR), ligated activated transcription (LAT) andnucleic acid sequence-based amplification (NASBA) may be used. Thenucleic acid sequence may be cloned from a strain of Geniculosporium, oranother or related organism and thus, for example, may be an allelic orspecies variant of the polypeptide encoding region of the nucleic acidsequence.

[0053] The term “isolated nucleic acid sequence” as used herein refersto a nucleic acid sequence which is essentially free of other nucleicacid sequences, e.g., at least about 20% pure, preferably at least about40% pure, more preferably at least about 60% pure, even more preferablyat least about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

[0054] The present invention also relates to nucleic acid sequenceswhich have a degree of homology to the polypeptide coding sequence ofSEQ ID NO:1 of at least about 80%, preferably about 90%, more preferablyabout 95%, and most preferably about 97% homology, which encode anactive polypeptide. For purposes of the present invention, the degree ofhomology between two nucleic acid sequences is determined by using GAPversion 8 from the GCG package with standard penalties for DNA: GAPweight 5.00, length weight 0.300, Matrix described in Gribskov andBurgess, Nucl. Acids Res. 14(16); 6745-6763 (1986).

[0055] Modification of a nucleic acid sequence encoding a polypeptide ofthe present invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant sequence may be constructed on the basis of thenucleic acid sequence presented as the polypeptide encoding part of SEQID NO:1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleic acid sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

[0056] It will be apparent to those skilled in the art that suchsubstitutions can be made outside the regions critical to the functionof the molecule and still result in an active polypeptide. Amino acidresidues essential to the activity of the polypeptide encoded by theisolated nucleic acid sequence of the invention, and thereforepreferably not subject to substitution, may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, mutations areintroduced at every positively charged residue in the molecule, and theresultant mutant molecules are tested for haloperoxidase activity toidentify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904;Wlodaver et al., 1992, FEBS Letters 309: 59-64).

[0057] The present invention also relates to isolated nucleic acidsequences encoding a polypeptide of the present invention, whichhybridize under low stringency conditions, preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with a nucleic acid probe which hybridizes underthe same conditions with the nucleic acid sequence of SEQ ID NO:1 or itscomplementary strand; or allelic variants and subsequences thereof(Sambrook et al., 1989, supra), as defined herein.

[0058] The present invention also relates to isolated nucleic acidsequences produced by (a) hybridizing a DNA under low, medium,medium-high, high, or very high stringency conditions with (i) thenucleotide sequence of SEQ ID NO:1, (ii) a subsequence of (i), or (iii)a complementary strand of (i), (ii) or (iii); and (b) isolating thenucleic acid sequence. The subsequence is preferably a sequence of atleast 100 nucleotides such as a sequence, which encodes a polypeptidefragment which has haloperoxidase activity.

Methods for Producing Mutant Nucleic Acid Sequences

[0059] The present invention further relates to methods for producing amutant nucleic acid sequence, comprising introducing at least onemutation into the polypeptide coding sequence of SEQ ID NO:1 or asubsequence thereof, wherein the mutant nucleic acid sequence encodes apolypeptide which consists of the amino acid sequence of SEQ ID NO:2 ora fragment thereof which has haloperoxidase activity.

[0060] The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure, which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with Dpnl which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used.

Nucleic Acid Constructs

[0061] The present invention also relates to nucleic acid constructscomprising a nucleic acid sequence of the present invention operablylinked to one or more control sequences, which direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences. Expression will be understood to include anystep involved in the production of the polypeptide including, but notlimited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.

[0062] “Nucleic acid construct” is defined herein as a nucleic acidmolecule, either single- or double-stranded, which is isolated from anaturally occurring gene or which has been modified to contain segmentsof nucleic acid combined and juxtaposed in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence of the present invention. The term “coding sequence” is definedherein as a nucleic acid sequence, which directly specifies the aminoacid sequence of its protein product. The boundaries of a genomic codingsequence are generally determined by a ribosome binding site(prokaryotes) or by the ATG start codon (eukaryotes) located justupstream of the open reading frame at the 5′ end of the mRNA and atranscription terminator sequence located just downstream of the openreading frame at the 3′ end of the mRNA. A coding sequence can include,but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

[0063] An isolated nucleic acid sequence encoding a polypeptide of thepresent invention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

[0064] The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolypeptide of the present invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide. The term “operably linked” is definedherein as a configuration in which a control sequence is appropriatelyplaced at a position relative to the coding sequence of the DNA sequencesuch that the control sequence directs the expression of a polypeptide.

[0065] The control sequence may be an appropriate promoter sequence, anucleic acid sequence that is recognized by a host cell for expressionof the nucleic acid sequence. The promoter sequence containstranscriptional control sequences, which mediate the expression of thepolypeptide. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

[0066] Examples of suitable promoters for directing the transcription ofthe nucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coil lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

[0067] Examples of suitable promoters for directing the transcription ofthe nucleic acid constructs of the present invention in a filamentousfungal host cell are promoters obtained from the genes for Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787),as well as the NA2-tpi promoter (a hybrid of the promoters from thegenes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

[0068] In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

[0069] The control sequence may also be a suitable transcriptionterminator sequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

[0070] Preferred terminators for filamentous fungal host cells areobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease.

[0071] Preferred terminators for yeast host cells are obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

[0072] The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

[0073] Preferred leaders for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase and Aspergillusnidulans triose phosphate isomerase.

[0074] Suitable leaders for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

[0075] The control sequence may also be a polyadenylation sequence, asequence operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

[0076] Preferred polyadenylation sequences for filamentous fungal hostcells are obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

[0077] Useful polyadenylation sequences for yeast host cells aredescribed by Guo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.

[0078] The control sequence may also be a signal peptide coding regionthat codes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

[0079] Effective signal peptide coding regions for bacterial host cellsare the signal peptide coding regions obtained from the genes forBacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0080] Effective signal peptide coding regions for filamentous fungalhost cells are the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

[0081] Useful signal peptides for yeast host cells are obtained from thegenes for Saccharomyces cerevisiae alpha-factor and Saccharomycescerevisiae invertase. Other useful signal peptide coding regions aredescribed by Romanos et al., 1992, supra.

[0082] The control sequence may also be a propeptide coding region thatcodes for an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

[0083] Where both signal peptide and propeptide regions are present atthe amino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

[0084] It may also be desirable to add regulatory sequences, which allowthe regulation of the expression of the polypeptide relative to thegrowth of the host cell. Examples of regulatory systems are those whichcause the expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GALL systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

[0085] The present invention also relates to recombinant expressionvectors comprising a nucleic acid sequence of the present invention, apromoter, and transcriptional and translational stop signals. Thevarious nucleic acid and control sequences described above may be joinedtogether to produce a recombinant expression vector which may includeone or more convenient restriction sites to allow for insertion orsubstitution of the nucleic acid sequence encoding the polypeptide atsuch sites. Alternatively, the nucleic acid sequence of the presentinvention may be expressed by inserting the nucleic acid sequence or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

[0086] The recombinant expression vector may be any vector (e.g., aplasmid or virus), which can be conveniently subjected to recombinantDNA procedures and can bring about the expression of the nucleic acidsequence. The choice of the vector will typically depend on the Itcompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

[0087] The vector may be an autonomously replicating vector, ie., avector which, exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

[0088] The vectors of the present invention preferably contain one ormore selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

[0089] The vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

[0090] For integration into the host cell genome, the vector may rely onthe nucleic acid sequence encoding the polypeptide or any other elementof the vector for integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 10,000 base pairs, preferably 400 to10,000 base pairs, and most preferably 800 to 10,000 base pairs, whichare highly homologous with the corresponding target sequence to enhancethe probability of homologous recombination. The integrational elementsmay be any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

[0091] For autonomous replication, the vector may further comprise anorigin of replication enabling the vector to replicate autonomously inthe host cell in question. Examples of bacterial origins of replicationare the origins of replication of plasmids pBR322, pUC19, pACYC177, andpACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060,and pAMβ1 permitting replication in Bacillus. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6. The origin of replication may be onehaving a mutation which makes its functioning temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proceedings of the NationalAcademy of Sciences USA 75: 1433).

[0092] More than one copy of a nucleic acid sequence of the presentinvention may be inserted into the host cell to increase production ofthe gene product. An increase in the copy number of the nucleic acidsequence can be obtained by integrating at least one additional copy ofthe sequence into the host cell genome or by including an amplifiableselectable marker gene with the nucleic acid sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleic acid sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent.

[0093] The procedures used to ligate the elements described above toconstruct the recombinant expression vectors of the present inventionare well known to one skilled in the art (see, e.g., Sambrook et al.,1989, supra).

Host Cells

[0094] The present invention also relates to recombinant host cells,comprising a nucleic acid sequence of the invention, which areadvantageously used in the recombinant production of the polypeptides. Avector comprising a nucleic acid sequence of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

[0095] The host cell may be a unicellular microorganism, e.g., aprokaryote, or a non-unicellular microorganism, e.g., a eukaryote.

[0096] Useful unicellular cells are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred embodiment, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred embodiment, the Bacilluscell is an alkalophilic Bacillus.

[0097] The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

[0098] The host cell may be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

[0099] In a preferred embodiment, the host cell is a fungal cell.“Fungi” as used herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

[0100] In a more preferred embodiment, the fungal host cell is a yeastcell. “Yeast” as used herein includes ascosporogenous yeast(Endomycetales), basidiosporogenous yeast, and yeast belonging to theFungi Imperfecti (Blastomycetes). Since the classification of yeast maychange in the future, for the purposes of this invention, yeast shall bedefined as described in Biology and Activities of Yeast (Skinner, F. A.,Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol.Symposium Series No. 9, 1980).

[0101] In an even more preferred embodiment, the yeast host cell is aCandida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia cell.

[0102] In a most preferred embodiment, the yeast host cell is aSaccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis cell. In another mostpreferred embodiment, the yeast host cell is a Kluyveromyces lactiscell. In another most preferred embodiment, the yeast host cell is aYarrowia lipolytica cell.

[0103] In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are generally characterizedby a mycelial wall composed of chitin, cellulose, glucan, chitosan,mannan, and other complex polysaccharides. Vegetative growth is byhyphal elongation and carbon catabolism is obligately aerobic. Incontrast, vegetative growth by yeasts such as Saccharomyces cerevisiaeis by budding of a unicellular thallus and carbon catabolism may befermentative.

[0104] In an even more preferred embodiment, the filamentous fungal hostcell is a cell of a species of, but not limited to, Acremonium,Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora,Penicillium, Thielavia, Tolypocladium, or Trichoderma.

[0105] In a most preferred embodiment, the filamentous fungal host cellis an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal parent cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0106] Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Methods of Production

[0107] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide, to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide. Preferably, the strain is of the genusGeniculosporium.

[0108] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

[0109] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleic acid sequence having atleast one mutation in the polypeptide coding region of SEQ ID NO:1,wherein the mutant nucleic acid sequence encodes a polypeptide whichconsists of the amino acid sequence of SEQ ID NO:2, and (b) recoveringthe polypeptide.

[0110] In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, and small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

[0111] The polypeptides may be detected using methods known in the artthat are specific for the polypeptides. These detection methods mayinclude use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate. For example, an enzyme assay maybe used to determine the activity of the polypeptide as describedherein.

[0112] The resulting polypeptide may be recovered by methods known inthe art. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

[0113] The polypeptides of the present invention may be purified by avariety of procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J. -C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Plants

[0114] The present invention also relates to a transgenic plant, plantpart, or plant cell, which has been transformed with a nucleic acidsequence encoding a polypeptide having haloperoxidase activity of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing therecombinant polypeptide may be used as such for improving the quality ofa food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

[0115] The transgenic plant can be dicotyledonous (a dicot) ormonocotyledonous (a monocot). Examples of monocot plants are grasses,such as meadow grass (blue grass, Poa), forage grass such as festuca,lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat,oats, rye, barley, rice, sorghum, and maize (corn).

[0116] Examples of dicot plants are tobacco, legumes, such as lupins,potato, sugar beet, pea, bean and soybean, and cruciferous plants(family Brassicaceae), such as cauliflower, rape seed, and the closelyrelated model organism Arabidopsis thaliana.

[0117] Examples of plant parts are stem, callus, leaves, root, fruits,seeds, and tubers. Also specific plant tissues, such as chloroplast,apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm areconsidered to be a plant part. Furthermore, any plant cell, whatever thetissue origin, is considered to be a plant part.

[0118] Also included within the scope of the present invention are theprogeny of such plants, plant parts and plant cells.

[0119] The transgenic plant or plant cell expressing a polypeptide ofthe present invention may be constructed in accordance with methodsknown in the art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

[0120] Conveniently, the expression construct is a nucleic acidconstruct, which comprises a nucleic acid sequence encoding apolypeptide of the present invention operably linked with appropriateregulatory sequences required for expression of the nucleic acidsequence in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying host cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

[0121] The choice of regulatory sequences, such as promoter andterminator sequences and optionally signal or transit sequences isdetermined, for example, on the basis of when, where, and how thepolypeptide is desired to be expressed. For instance, the expression ofthe gene encoding a polypeptide of the present invention may beconstitutive or inducible, or may be developmental, stage or tissuespecific, and the gene product may be targeted to a specific tissue orplant part such as seeds or leaves. Regulatory sequences are, forexample, described by Tague et al., 1988, Plant Physiology 86: 506.

[0122] For constitutive expression, the 35S-CaMV promoter may be used(Franck et al., 1980, Cell 21: 285-294). Organ-specific promoters maybe, for example, a promoter from storage sink tissues such as seeds,potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588).

[0123] A promoter enhancer element may also be used to achieve higherexpression of the enzyme in the plant. For instance, the promoterenhancer element may be an intron, which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

[0124] The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

[0125] The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

[0126] Presently, Agrobacterium tumefaciens-mediated gene transfer isthe method of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

[0127] Following transformation, the transformants having incorporatedtherein the expression construct are selected and regenerated into wholeplants according to methods well-known in the art.

[0128] The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having haloperoxidase activity of the presentinvention under conditions conducive for production of the polypeptide;and (b) recovering the polypeptide.

Compositions

[0129] In a still further aspect, the present invention relates tocompositions comprising a polypeptide of the present invention.Preferably, the compositions are enriched in a polypeptide of thepresent invention. In the present context, the term “enriched” indicatesthat the haloperoxidase activity of the composition has been increased,e.g., with an enrichment factor of 1.1.

[0130] The composition may comprise a polypeptide of the invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be producible by means of amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus niger, or Aspergillusoryzae, or Trichoderma, Humicola, preferably Humicola insolens, orFusarium, preferably Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusariumtrichothecioides, or Fusarium venenatum.

[0131] The polypeptide compositions may be prepared in accordance withmethods known in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

[0132] Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Detergent Composition

[0133] The haloperoxidase of the invention may be added to and thusbecome a component of a detergent composition.

[0134] The detergent composition of the invention may for example beformulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or be formulatedas a detergent composition for use in general household hard surfacecleaning operations, or be formulated for hand or machine dishwashingoperations.

[0135] In a specific aspect, the invention provides a detergent additivecomprising the haloperoxidase of the invention. The detergent additiveas well as the detergent composition may comprise one or more otherenzymes such as a protease, a lipase, a cutinase, an amylase, acarbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase, e.g., a laccase, and/or aperoxidase.

[0136] In general the properties of the chosen enzyme(s) should becompatible with the selected detergent, (i.e. pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzyme(s) should be present in effective amounts.

Proteases

[0137] Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g. of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

[0138] Examples of useful proteases are the variants described in WO92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially thevariants with substitutions in one or more of the following positions:27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218,222, 224, 235 and 274.

[0139] Preferred commercially available protease enzymes includeAlcalase™, Savinase™, Primase™, Everlase™, Esperase™, and Kannase™(Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™,Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.).

Lipases

[0140] Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. fromB. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131,253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

[0141] Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

[0142] Preferred commercially available lipase enzymes include Lipolase™and Lipolase Ultra™ (Novozymes A/S).

Amylases

[0143] Suitable amylases (α and/or β) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, α-amylases obtained fromBacillus, e.g. a special strain of B. licheniformis, described in moredetail in GB 1,296,839.

[0144] Examples of useful amylases are the variants described in WO94/02597, WO 94/18314, WO 96/23873, and WO 97143424, especially thevariants with substitutions in one or more of the following positions:15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208,209, 243, 264, 304, 305, 391, 408, and 444.

[0145] Commercially available amylases are Duramyl™, Termamyl™,Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™ (fromGenencor International Inc.).

Cellulases

[0146] Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263,U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

[0147] Especially suitable cellulases are the alkaline or neutralcellulases having colour care benefits. Examples of such cellulases arecellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO96/29397, WO 98/08940. Other examples are cellulase variants such asthose described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046,U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO98/12307 and PCT/DK98/00299.

[0148] Commercially available cellulases include Celluzyme™, andCarezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases

[0149] Suitable peroxidases/oxidases include those of plant, bacterialor fungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g. from C. cinereus, and variants thereof as those describedin WO 93/24618, WO 95/10602, and WO 98/15257.

[0150] Commercially available peroxidases include Guardzyme™ (NovozymesA/S).

[0151] The detergent enzyme(s) may be included in a detergentcomposition by adding separate additives containing one or more enzymes,or by adding a combined additive comprising all of these enzymes. Adetergent additive of the invention, i.e. a separate additive or acombined additive, can be formulated e.g. as a granulate, a liquid, aslurry, etc. Preferred detergent additive formulations are granulates,in particular non-dusting granulates, liquids, in particular stabilizedliquids, or slurries.

[0152] Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. No. 4,106,991 and 4,661,452 and may optionally be coated bymethods known in the art. Examples of waxy coating materials arepoly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molarweights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50ethylene oxide units; ethoxylated fatty alcohols in which the alcoholcontains from 12 to 20 carbon atoms and in which there are 15 to 80ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

[0153] The detergent composition of the invention may be in anyconvenient form, e.g., a bar, a tablet, a powder, a granule, a paste ora liquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

[0154] The detergent composition comprises one or more surfactants,which may be non-ionic including semi-polar and/or anionic and/orcationic and/or zwitterionic. The surfactants are typically present at alevel of from 0.1% to 60% by weight.

[0155] When included therein the detergent will usually contain fromabout 1% to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

[0156] When included therein the detergent will usually contain fromabout 0.2% to about 40% of a non-ionic surfactant such as alcoholethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acylN-alkyl derivatives of glucosamine (“glucamides”).

[0157] The detergent may contain 0-65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g. SKS-6 from Hoechst).

[0158] The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

[0159] The detergent may contain a bleaching system, which may comprisea H₂O₂ source such as perborate or percarbonate which may be combinedwith a peracid-forming bleach activator such astetraacetylethylenediamine or nonanoyloxybenzenesulfonate.Alternatively, the bleaching system may comprise peroxyacids of e.g. theamide, imide, or sulfone type.

[0160] The enzyme(s) of the detergent composition of the invention maybe stabilized using conventional stabilizing agents, e.g., a polyol suchas propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

[0161] The detergent may also contain other conventional detergentingredients such as e.g. fabric conditioners including clays, foamboosters, suds suppressors, anti-corrosion agents, soil-suspendingagents, anti-soil redeposition agents, dyes, bactericides, opticalbrighteners, hydrotropes, tarnish inhibitors, or perfumes.

[0162] It is at present contemplated that in the detergent compositionsany enzyme, in particular the haloperoxidase of the invention, may beadded in an amount corresponding to 0.01-100 mg of enzyme protein perliter of wash liqour, preferably 0.05-5 mg of enzyme protein per literof wash liquor, in particular 0.1-1 mg of enzyme protein per liter ofwash liquor.

[0163] The haloperoxidase of the invention may additionally beincorporated in the detergent formulations disclosed in WO 97/07202,which is hereby incorporated as reference.

Uses

[0164] The present invention is also directed to methods for using thepolypeptides having haloperoxidase activity.

[0165] The present invention is further directed to methods of oxidizinga halide ion to the corresponding hypohalous acid, comprising reactingthe halide ion and a source of hydrogen peroxide in the presence of ahaloperoxidase of the invention. The present invention also relates tomethods of halogenating a compound comprising reacting the compound, ahalide ion and a source of hydrogen peroxide in the presence of ahaloperoxidase of the invention.

[0166] The present invention also relates to methods for killing orinhibiting the growth of microbial cells, comprising contacting thecells with a haloperoxidase of the invention, a source of hydrogenperoxide, and a source of halide or thiocyanate in an aqueous solution.

[0167] The source of hydrogen peroxide can be hydrogen peroxide itselfor a hydrogen peroxide precursor, such as, a percarbonate, perborate,peroxycarboxylic acid or a salt thereof. Furthermore, the source may bea hydrogen peroxide generating enzyme system, such as an oxidase, e.g.,a glucose oxidase, glycerol oxidase or amino acid oxidase, and itssubstrate. The hydrogen peroxide source may be added in a concentrationcorresponding to a hydrogen peroxide concentration in the range of fromabout 0.001 to about 10 mM, preferably about 0.01 to about 1 mM.

[0168] The halide source may be a halide salt, preferably a sodium orpotassium salt, such as sodium chloride, potassium chloride, sodiumbromide, potassium bromide, sodium iodide, or potassium iodide. Thethiocyanate source may be a thiocyanate salt, preferably a sodium orpotassium salt.

[0169] The concentration of the halide source will typically correspondto 0.001-1000 mM, preferably in the range of from 0.005-500 mM, morepreferably in the range of from 0.01-100 mM, and most preferably in therange of from 0.05-50 mM.

[0170] The haloperoxidases may be used as preservation agents anddisinfection agents such as in water based paints and personal careproducts, e.g., toothpaste, mouthwash, skin care creams and lotions,hair care and body care formulations, solutions for cleaning contactlenses and dentures. The haloperoxidases also may be used for cleaningsurfaces and cooking utensils in food processing plants and in any areain which food is prepared or served. The haloperoxidases also may beused in enzymatic bleaching applications, e.g., pulp bleaching and stainbleaching (in detergent compositions).

[0171] The concentration of the haloperoxidase in the methods of use ofthe present invention, is preferably in the range of 0.01-50 mg/I, morepreferably in the range of 0.1-10 mg/l.

DNA recombination shuffling

[0172] The nucleotide sequence of SEQ ID NO:1 may be used in a DNArecombination (or shuffling) process. The new polynucleotide sequencesobtained in such a process may encode new polypeptides havinghaloperoxidase activity with improved properties, such as improvedstability (storage stability, thermostability), improved specificactivity, improved pH-optimum, and/or improved tolerance towardsspecific compounds.

[0173] Shuffling between two or more homologous input polynucleotides(starting-point polynucleotides) involves fragmenting thepolynucleotides and recombining the fragments, to obtain outputpolynucleotides (i.e. polynucleotides that have been subjected to ashuffling cycle) wherein a number of nucleotide fragments are exchangedin comparison to the input polynucleotides.

[0174] DNA recombination or shuffling may be a (partially) randomprocess in which a library of chimeric genes is generated from two ormore starting genes. A number of known formats can be used to carry outthis shuffling or recombination process.

[0175] The process may involve random fragmentation of parental DNAfollowed by reassembly by PCR to new full-length genes, e.g. aspresented in U.S. Pat No. 5,605,793, U.S. Pat No. 5,811,238, U.S. Pat.No. 5,830,721, U.S. Pat. No. 6,117,679. In-vitro recombination of genesmay be carried out, e.g. as described in U.S. Pat. No. 6,159,687,WO98/41623, U.S. Pat. No. 6,159,688, U.S. Pat. No. 5,965,408, U.S. Pat.No. 6,153,510. The recombination process may take place in vivo in aliving cell, e.g. as described in WO 97/07205 and WO 98/28416.

[0176] The parental DNA may be fragmented by DNA'se I treatment or byrestriction endonuclease digests as descriobed by Kikuchi et al (2000a,Gene 236:159-167). Shuffling of two parents may be done by shufflingsingle stranded parental DNA of the two parents as described in Kikuchiet al (2000b, Gene 243:133-137).

[0177] A particular method of shuffling is to follow the methodsdescribed in Crameri et al, 1998, Nature, 391: 288-291 and Ness et al.Nature Biotechnology 17: 893-896. Another format would be the methodsdescribed in U.S. Pat. No. 6,159,687: Examples 1 and 2.

[0178] The present invention is further described by the followingexamples, which should not be construed as limiting the scope of theinvention.

EXAMPLES

[0179] Chemicals used as buffers and substrates were commercial productsof at least reagent grade.

Haloperoxidase assays

[0180] Microtiter assays are performed by mixing 100 μl ofhaloperoxidase sample (about 0.2 μg/ml) and 100 μl assay buffer (0.3 Msodium phosphate; pH 7; 1.25 mM Na₃VO₄; 50 mM KBr; 0.008% phenol red).Reactions were initiated by adding 10 μl of 0.3% H₂O₂, and theabsorption at 595 nm was measured spectrophotometrically as a functionof time in a Molecular Devices Kinetic Microplate reader.

[0181] Assays using monochlorodimedone (Sigma M4632, ε=20000 M⁻¹cm⁻¹ at290 nm) as a substrate are performed as described below. The decrease inabsorption at 290 nm is measured as a function of time. Assays areperformed in 0.1 M sodium phosphate or 0.1 M sodium acetate, 50 μMmonochlorodimedone, 10 mM KBr/KCl, and 1 mM H₂O₂ using a haloperoxidaseconcentration of about 1 μg/ml. One HU is defined as 1 micromol ofmonochlorodimedone chlorinated or brominated per minute at pH 5 and 30°C.

Example 1 Transformation and Fermentation of Aspergillus orvzae

[0182] Protoplast preparation and transformation in Aspergillus oryzaeof the nucleic acid sequence encoding the Geniculosporium sp.haloperoxidase contained in the plasmid contained in E. coli DH10B,deposited as DSM 13442, was done essentially as described by Christensenet al. (1988), Biotechnology, 6:1419-1422. Transformants were plated onAMDS agar plates selecting for the ability to grow on acetamide as solenitrogen source.

[0183]A. oryzae transformants were spore purified twice and inoculatedinto 100 μl YP growth medium supplemented with maltose (3%), 1 mMNa₃VO₄, and 0.4% Urea. Cultures were grown at 34° C. for 5 days afterwhich they were assayed for haloperoxidase activity. The besthaloperoxidase producer from the transformation was inoculated into8×125 ml baffled flasks containing YP growth medium with maltose (3%), 1mM Na₃VO₄, and 0.4% Urea and grown for 7 days at 34° C., with shaking at200 rpm.

Example 2 Purification of Geniculosporium sp. haloperoxidase

[0184] Glucanex™ (available from Novozymes A/S) treated fermentationbroth was centrifuged and the supernatant was filtered through a SeitzEKS filter plate (Seitz-Filter-Werke GmbH, Germany) and concentrated andwashed by ultrafiltration on a Filtron Minisette™ system (FiltronTechnology Corporation, Massachusetts, USA) with an Omega type membrane(Mw cut-off of 10 kDa) to a final volume of 300 ml and a conductanceless than 2 mS.

[0185] The crude enzyme preparation was filtered on a glass filter,added 6 ml of a 10 mM sodium orthovanadate, and applied onto an anionexchange column (Pharmacia 26/10 with Q-Sepharose High Performance)equilibrated with 50 mM Tris/HCl pH 7. The column was washed withequilibration buffer and eluted with a 0-1M sodium chloride gradient(over 10 column volumes) in the same buffer using a flow of 10ml/minute. Fractions of 10 ml were collected and tested forhaloperoxidase activity.

[0186] Fractions showing haloperoxidase activity were pooled,concentrated by ultrafiltration on an Amicon cell (membrane Mw cut-offof 10 kDa) to a final volume of 6-7 ml. Two times 2 ml of theconcentrated sample was applied onto a gel filtration column (PharmaciaHiLoad 16/60, Su-perdex 200 High Performance) equilibrated with 50 mMsodium acetate, 100 mM NaCl pH 5.5. The column was eluted with a flow of1 ml/minute and fractions of 1 ml were collected. Fractions from bothruns showing HPO activity were pooled giving 22 ml with A280=10.766. Thepooled fractions contained rather pure haloperoxidase showing only oneband on SDS-PAGE with a Mr close to 65 kDa.

Example 3 Thermal Stability of Recombinant Geniculosporium sp.haloperoxidase

[0187] The thermal stability of Geniculosporium sp. haloperoxidase wasdetermined by the following procedure:

[0188] The enzyme was diluted to an absorbance at 280 nm of approx. 0.1in a 0.3 M Tris/HCl buffer pH 7. 0.5 ml portions of the dilution wasincubated at 30, 40, 50, 60, 70, and 80° C., respectively, for 15minutes and then placed on ice-water. A reference sample of the dilutedenzyme was kept at 4° C. Activity of the samples was measured accordingto the phenol red assay in microwell plate using bromide as substrate,and residual activity was calculated relatively to the reference (storedat 4° C.).

Conclusion

[0189] The residual activity of Geniculosporium sp. haloperoxidase is atleast 60% after 15 minutes incubation at 70° C. TABLE 1 Temperature (°C.) Residual activity (%) 4 100 30 103.3 40 100.2 50 92.8 60 84.5 7061.9 80 14.6

Example 4 Antibacterial Activity of Geniculosporium sp. haloperoxidaseAgainst Escherichia coli

[0190] The antibacterial activity of the Geniculosporium sp.haloperoxidase, available from Novozymes A/S, DK-2880 Bagsvaerd,Denmark, was tested with bromide as enhancing agent.

[0191] The antibacterial activity of haloperoxidase was tested inHEPES-buffer (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonicacid])(pH 7.0) against Escherichia coli DSM 1576 with potassium bromideas electron donor, and hydrogen peroxide was added as electron acceptor.The cells (approximately 10⁶ CFU/ml) were incubated with enzyme for 15min at 40° C.

[0192] The bactericidal activity was determined by incubation in aMalthus Flexi M2060 instrument (available from Malthus InstrumentsLimited, England). The detection times measured by the Malthusinstrument were converted to CFU/ml (colony forming units pr. ml) by acalibration curve. Direct measurements were used when enumerating totalsurvival cells. By the direct measurements, the cell metabolism wasdetermined by conductance measurements in the growth substrate. When theconductance change is measurable by the Malthus instrument, a detectiontime (dt) will be recorded. The dt's were converted to colony counts byuse of a calibration curve relating CFU/ml to dt.

Results

[0193] TABLE 2 KBr Enzyme H₂O₂ Bactericidal activity (mM) (mg/L) (mM)(log CFU/ml) (doublets) 0 0 0 −0.1/0.1  8 0 0 0.3/0.4 0 0 1 0.2/0.8 8 01 0.6/0.5 0 1 0 0.4/0.4 8 1 1 6.4*/6.4*

[0194] Bactericidal activity is shown in the table as log₁₀ reduction inthe number of living cells (colony forming units), thus a bactericidalactivity of 6 correspond to a kill of 10⁶ CFU/ml. A significantbactericidal activity was obtained with the Geniculosporium sp.haloperoxidase, and no significant bactericidal activity was obtainedwith any of the controls.

DEPOSIT OF BIOLOGICAL MATERIAL

[0195] An E. Coli DH10B clone containing a haloperoxidase gene fromGeniculosporium sp. (SEQ ID NO:1) inserted into a pUC19 derived plasmidhas been deposited under the terms of the Budapest Treaty with theDeutsche Sammlung von Mikroorganismen und Zelikulturen GmbH (DSMZ),Mascheroder Weg 1b, D-38124 Braunschweig, Germany, and given thefollowing accession number: Deposit Accession Number Date of DepositNN049533 DSM 13442 2000-Apr-12

[0196] The deposit was made by Novo Nordisk A/S and was later assignedto Novozymes A/S. The strain has been deposited under conditions thatassure that access to the culture will be available during the pendencyof this patent application to one determined by the Commissioner ofPatents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and35 U.S.C. §122. The deposit represents a substantially pure culture ofthe deposited strain. The deposit is available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

[0197] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims. In the case of conflict, the present disclosure includingdefinitions will control.

[0198] Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1 2 1 1842 DNA Geniculosporium sp. CDS (1)..(1842) 1 atg gca aca ttc acccct atc ccg ctt cct caa att gat gaa ccg gca 48 Met Ala Thr Phe Thr ProIle Pro Leu Pro Gln Ile Asp Glu Pro Ala 1 5 10 15 gag tac aac acg aactac gtt ctg tat tgg cac cat gtc ggc ttg gag 96 Glu Tyr Asn Thr Asn TyrVal Leu Tyr Trp His His Val Gly Leu Glu 20 25 30 ctt aac cgc gtc aca catacc gtc gga ggc ccg cag act ggc cca cca 144 Leu Asn Arg Val Thr His ThrVal Gly Gly Pro Gln Thr Gly Pro Pro 35 40 45 atc tct gct aga gct ctg ggcatg ctc cag ttg gct gta cat gat gcc 192 Ile Ser Ala Arg Ala Leu Gly MetLeu Gln Leu Ala Val His Asp Ala 50 55 60 tac ttc gcc atc cat ccc tct tctagc ttc ctg acc ttt ttg aca tct 240 Tyr Phe Ala Ile His Pro Ser Ser SerPhe Leu Thr Phe Leu Thr Ser 65 70 75 80 ggc gcc gac aac cct gcc tac gctctg ccc gag ttg agc ggc gcg gac 288 Gly Ala Asp Asn Pro Ala Tyr Ala LeuPro Glu Leu Ser Gly Ala Asp 85 90 95 gat gcc cgc cag gcg gta gct ggt gcatct gtt act atg ctg tct atg 336 Asp Ala Arg Gln Ala Val Ala Gly Ala SerVal Thr Met Leu Ser Met 100 105 110 ctt tac atg aag ccc cct acc aac cccaac ccc aat cct ggc gcc acc 384 Leu Tyr Met Lys Pro Pro Thr Asn Pro AsnPro Asn Pro Gly Ala Thr 115 120 125 att tcc gac aac gcc tat gca cag cttcag tat gtc att gac aaa tca 432 Ile Ser Asp Asn Ala Tyr Ala Gln Leu GlnTyr Val Ile Asp Lys Ser 130 135 140 gta acc gat gca ccc ggt ggt gta gatgca gcg tcc agc agt ttc aac 480 Val Thr Asp Ala Pro Gly Gly Val Asp AlaAla Ser Ser Ser Phe Asn 145 150 155 160 ttc gga aag gca gta gct act gtcttc ttc aac cta ctc ttc cac gcc 528 Phe Gly Lys Ala Val Ala Thr Val PhePhe Asn Leu Leu Phe His Ala 165 170 175 ccg ggt gcc tca caa gct ggc tatcac cct aca ccc ggc cca tac aag 576 Pro Gly Ala Ser Gln Ala Gly Tyr HisPro Thr Pro Gly Pro Tyr Lys 180 185 190 ttc gac gat gag ccc act cac cctgtc gtc ctt gtt ccc gtt gac gca 624 Phe Asp Asp Glu Pro Thr His Pro ValVal Leu Val Pro Val Asp Ala 195 200 205 aac aac ccg gat ggt ccc aag cggcct ttc cgc cag tat cac ggc ccg 672 Asn Asn Pro Asp Gly Pro Lys Arg ProPhe Arg Gln Tyr His Gly Pro 210 215 220 ttc tat ggc aag act gct aag cgtttt gct aca cag act gag cat atg 720 Phe Tyr Gly Lys Thr Ala Lys Arg PheAla Thr Gln Thr Glu His Met 225 230 235 240 att gct gac ccg cca gcc attcgt tct gcc gtt ggt gag caa gct gaa 768 Ile Ala Asp Pro Pro Ala Ile ArgSer Ala Val Gly Glu Gln Ala Glu 245 250 255 tac gat gat agt att cgt caaatc att gcc atg ggt gga gct acc ggc 816 Tyr Asp Asp Ser Ile Arg Gln IleIle Ala Met Gly Gly Ala Thr Gly 260 265 270 ctc aac tcc acc aag cgc agccct ttt cag aca act caa ggc atg ttc 864 Leu Asn Ser Thr Lys Arg Ser ProPhe Gln Thr Thr Gln Gly Met Phe 275 280 285 tgg gcc tac gat ggc tcc aacttg gtc ggc aca cca ccc aga ttt tac 912 Trp Ala Tyr Asp Gly Ser Asn LeuVal Gly Thr Pro Pro Arg Phe Tyr 290 295 300 aac cag att gtc cgc cgc atcgca gtg acg tac aag aag gag gaa gac 960 Asn Gln Ile Val Arg Arg Ile AlaVal Thr Tyr Lys Lys Glu Glu Asp 305 310 315 320 ttg act aac agc gaa gtcaac aac gca gac ttt gtc cgt ctc ctt gct 1008 Leu Thr Asn Ser Glu Val AsnAsn Ala Asp Phe Val Arg Leu Leu Ala 325 330 335 ctg gtc aac gta gcc tgtgcc gat gca gga atc ttc tcc tgg aaa gag 1056 Leu Val Asn Val Ala Cys AlaAsp Ala Gly Ile Phe Ser Trp Lys Glu 340 345 350 aag tgg gaa ttt gaa ttctgg cgc cca ctc tct ggt gtt cgt gac gac 1104 Lys Trp Glu Phe Glu Phe TrpArg Pro Leu Ser Gly Val Arg Asp Asp 355 360 365 aac ttc cgc gac cca aatcgc cca gat cgt ggc gac cct ttc tgg ctt 1152 Asn Phe Arg Asp Pro Asn ArgPro Asp Arg Gly Asp Pro Phe Trp Leu 370 375 380 act ctc ggc gcc cca gccaca aac aca aac gac att cct ttc aaa ccc 1200 Thr Leu Gly Ala Pro Ala ThrAsn Thr Asn Asp Ile Pro Phe Lys Pro 385 390 395 400 ccc ttc ccc gct tacccc tct ggt cac gcc aca ttc ggt ggc gcc gtc 1248 Pro Phe Pro Ala Tyr ProSer Gly His Ala Thr Phe Gly Gly Ala Val 405 410 415 ttc cag atg gtc cgccgc tac tac aac ggg cga gtt gga aac tgg aaa 1296 Phe Gln Met Val Arg ArgTyr Tyr Asn Gly Arg Val Gly Asn Trp Lys 420 425 430 gac gac gaa gtg gacaac atc gcc atc gat atg atg gta tcc gag gag 1344 Asp Asp Glu Val Asp AsnIle Ala Ile Asp Met Met Val Ser Glu Glu 435 440 445 ctc aac ggg ttg agccgt gat ctc cgc caa ccc tac gac ccc aaa gcg 1392 Leu Asn Gly Leu Ser ArgAsp Leu Arg Gln Pro Tyr Asp Pro Lys Ala 450 455 460 ccc att acc gat cagcca ggt atc gtg cgc aca cga gtt cca cgc cac 1440 Pro Ile Thr Asp Gln ProGly Ile Val Arg Thr Arg Val Pro Arg His 465 470 475 480 ttc tct tcc gtctgg gag atg atg ttc gag aac gca atc tcg cgt atc 1488 Phe Ser Ser Val TrpGlu Met Met Phe Glu Asn Ala Ile Ser Arg Ile 485 490 495 ttt ctc ggc gtccac tgg cgc ttc gat gct gca gcc gcc aag gat att 1536 Phe Leu Gly Val HisTrp Arg Phe Asp Ala Ala Ala Ala Lys Asp Ile 500 505 510 ttg atc ccc acgacg aca aag gat gtc tac gct gta gac aac aac ggc 1584 Leu Ile Pro Thr ThrThr Lys Asp Val Tyr Ala Val Asp Asn Asn Gly 515 520 525 gct tcc ttg ttccaa aac gtc gag gat att cgt tat acg act atg ggt 1632 Ala Ser Leu Phe GlnAsn Val Glu Asp Ile Arg Tyr Thr Thr Met Gly 530 535 540 act agg gag ggtcac gat ggg ctt ttg ccg att ggt ggt gtg ccg ctt 1680 Thr Arg Glu Gly HisAsp Gly Leu Leu Pro Ile Gly Gly Val Pro Leu 545 550 555 560 ggt att gggatt gcg aat gag atc ttt gat aca ggt ctc aag cct acc 1728 Gly Ile Gly IleAla Asn Glu Ile Phe Asp Thr Gly Leu Lys Pro Thr 565 570 575 cca ccg gagaaa cag cca gtg ccg ccg cct cca ttc aac cag agc gga 1776 Pro Pro Glu LysGln Pro Val Pro Pro Pro Pro Phe Asn Gln Ser Gly 580 585 590 cct acg aaggag atg ttg gag gaa gcg gga agt gag gag cag gtc cct 1824 Pro Thr Lys GluMet Leu Glu Glu Ala Gly Ser Glu Glu Gln Val Pro 595 600 605 atg atg gacgtt gcg ccc 1842 Met Met Asp Val Ala Pro 610 2 614 PRT Geniculosporiumsp. 2 Met Ala Thr Phe Thr Pro Ile Pro Leu Pro Gln Ile Asp Glu Pro Ala 15 10 15 Glu Tyr Asn Thr Asn Tyr Val Leu Tyr Trp His His Val Gly Leu Glu20 25 30 Leu Asn Arg Val Thr His Thr Val Gly Gly Pro Gln Thr Gly Pro Pro35 40 45 Ile Ser Ala Arg Ala Leu Gly Met Leu Gln Leu Ala Val His Asp Ala50 55 60 Tyr Phe Ala Ile His Pro Ser Ser Ser Phe Leu Thr Phe Leu Thr Ser65 70 75 80 Gly Ala Asp Asn Pro Ala Tyr Ala Leu Pro Glu Leu Ser Gly AlaAsp 85 90 95 Asp Ala Arg Gln Ala Val Ala Gly Ala Ser Val Thr Met Leu SerMet 100 105 110 Leu Tyr Met Lys Pro Pro Thr Asn Pro Asn Pro Asn Pro GlyAla Thr 115 120 125 Ile Ser Asp Asn Ala Tyr Ala Gln Leu Gln Tyr Val IleAsp Lys Ser 130 135 140 Val Thr Asp Ala Pro Gly Gly Val Asp Ala Ala SerSer Ser Phe Asn 145 150 155 160 Phe Gly Lys Ala Val Ala Thr Val Phe PheAsn Leu Leu Phe His Ala 165 170 175 Pro Gly Ala Ser Gln Ala Gly Tyr HisPro Thr Pro Gly Pro Tyr Lys 180 185 190 Phe Asp Asp Glu Pro Thr His ProVal Val Leu Val Pro Val Asp Ala 195 200 205 Asn Asn Pro Asp Gly Pro LysArg Pro Phe Arg Gln Tyr His Gly Pro 210 215 220 Phe Tyr Gly Lys Thr AlaLys Arg Phe Ala Thr Gln Thr Glu His Met 225 230 235 240 Ile Ala Asp ProPro Ala Ile Arg Ser Ala Val Gly Glu Gln Ala Glu 245 250 255 Tyr Asp AspSer Ile Arg Gln Ile Ile Ala Met Gly Gly Ala Thr Gly 260 265 270 Leu AsnSer Thr Lys Arg Ser Pro Phe Gln Thr Thr Gln Gly Met Phe 275 280 285 TrpAla Tyr Asp Gly Ser Asn Leu Val Gly Thr Pro Pro Arg Phe Tyr 290 295 300Asn Gln Ile Val Arg Arg Ile Ala Val Thr Tyr Lys Lys Glu Glu Asp 305 310315 320 Leu Thr Asn Ser Glu Val Asn Asn Ala Asp Phe Val Arg Leu Leu Ala325 330 335 Leu Val Asn Val Ala Cys Ala Asp Ala Gly Ile Phe Ser Trp LysGlu 340 345 350 Lys Trp Glu Phe Glu Phe Trp Arg Pro Leu Ser Gly Val ArgAsp Asp 355 360 365 Asn Phe Arg Asp Pro Asn Arg Pro Asp Arg Gly Asp ProPhe Trp Leu 370 375 380 Thr Leu Gly Ala Pro Ala Thr Asn Thr Asn Asp IlePro Phe Lys Pro 385 390 395 400 Pro Phe Pro Ala Tyr Pro Ser Gly His AlaThr Phe Gly Gly Ala Val 405 410 415 Phe Gln Met Val Arg Arg Tyr Tyr AsnGly Arg Val Gly Asn Trp Lys 420 425 430 Asp Asp Glu Val Asp Asn Ile AlaIle Asp Met Met Val Ser Glu Glu 435 440 445 Leu Asn Gly Leu Ser Arg AspLeu Arg Gln Pro Tyr Asp Pro Lys Ala 450 455 460 Pro Ile Thr Asp Gln ProGly Ile Val Arg Thr Arg Val Pro Arg His 465 470 475 480 Phe Ser Ser ValTrp Glu Met Met Phe Glu Asn Ala Ile Ser Arg Ile 485 490 495 Phe Leu GlyVal His Trp Arg Phe Asp Ala Ala Ala Ala Lys Asp Ile 500 505 510 Leu IlePro Thr Thr Thr Lys Asp Val Tyr Ala Val Asp Asn Asn Gly 515 520 525 AlaSer Leu Phe Gln Asn Val Glu Asp Ile Arg Tyr Thr Thr Met Gly 530 535 540Thr Arg Glu Gly His Asp Gly Leu Leu Pro Ile Gly Gly Val Pro Leu 545 550555 560 Gly Ile Gly Ile Ala Asn Glu Ile Phe Asp Thr Gly Leu Lys Pro Thr565 570 575 Pro Pro Glu Lys Gln Pro Val Pro Pro Pro Pro Phe Asn Gln SerGly 580 585 590 Pro Thr Lys Glu Met Leu Glu Glu Ala Gly Ser Glu Glu GlnVal Pro 595 600 605 Met Met Asp Val Ala Pro 610

1. An isolated nucleic acid sequence comprising a nucleic acid sequencewhich encodes a polypeptide having haloperoxidase activity, wherein thepolypeptide is selected from the group consisting of: (a) a polypeptidehaving an amino acid sequence which has at least 80% homology with theamino acid sequence of SEQ ID NO:2; (b) a polypeptide which is encodedby a nucleic acid sequence which hybridizes under medium stringencyconditions with (i) the nucleotide sequence of SEQ ID NO:1, (ii) asubsequence of (i) of at least 100 nucleotides, or (iii) a complementarystrand of (i) or (ii); (c) a variant of the polypeptide having an aminoacid sequence of SEQ ID NO:2 comprising a substitution, deletion, and/orinsertion of one or more amino acids; (d) an allelic variant of (a) or(b); (e) a fragment of (a), (b), or (d) that has haloperoxidaseactivity; and (f) a polypeptide having more than 50% residual activityafter 15 minutes incubation at 70° C. and pH
 7. 2. The nucleic acidsequence of claim 1, wherein the amino acid sequence of the polypeptidehas at least 95% homology with the amino acid sequence of SEQ ID NO:2.3. The nucleic acid sequence of claim 2, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:2.
 4. The nucleic acidsequence of claim 1, wherein the polypeptide consists of the amino acidsequence of SEQ ID NO:2 or a fragment thereof.
 5. The nucleic acidsequence of claim 4, wherein the polypeptide consists of the amino acidsequence of SEQ ID NO:2.
 6. The nucleic acid sequence of claim 1,wherein the polypeptide is a variant of the polypeptide having an aminoacid sequence of SEQ ID NO:2 comprising a substitution, deletion, and/orinsertion of one or more amino acids.
 7. The nucleic acid sequence ofclaim 1, which is contained in the plasmid contained in E. coli DH10B,deposited as DSM
 13442. 8. An isolated nucleic acid sequence comprisinga nucleic acid sequence having at least one mutation in the polypeptidecoding sequence of SEQ ID NO:1, in which the mutant nucleic acidsequence encodes a polypeptide consisting of the amino acid sequence ofSEQ ID NO:2.
 9. A nucleic acid construct comprising the nucleic acidsequence of any of claims 1-8 operably linked to one or more controlsequences that direct the production of the polypeptide in a suitableexpression host.
 10. A recombinant expression vector comprising thenucleic acid construct of claim
 9. 11. A recombinant host cellcomprising the nucleic acid construct of claim
 9. 12. A method forproducing a mutant nucleic acid sequence, comprising (a) introducing atleast one mutation into the polypeptide coding sequence of SEQ ID NO:1,wherein the mutant nucleic acid sequence encodes a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2; and (b) recoveringthe mutant nucleic acid sequence.
 13. A mutant nucleic acid sequenceproduced by the method of claim
 12. 14. A method for producing apolypeptide, comprising (a) cultivating a strain comprising the mutantnucleic acid sequence of claim 13 encoding the polypeptide to produce asupernatant comprising the polypeptide; and (b) recovering thepolypeptide.
 15. A method for producing the polypeptide comprising (a)cultivating a host cell comprising a nucleic acid construct comprising anucleic acid sequence of any of claims 1-8 encoding the polypeptideunder conditions suitable for production of the polypeptide; and (b)recovering the polypeptide.
 16. A method for producing a polypeptidecomprising (a) cultivating a host cell under conditions conducive forproduction of the polypeptide, wherein the host cell comprises a mutantnucleic acid sequence having at least one mutation in the polypeptidecoding sequence of SEQ ID NO:1, wherein the mutant nucleic acid sequenceencodes a polypeptide consisting of the amino acid sequence of SEQ IDNO:2, and (b) recovering the polypeptide.
 17. A method for shuffling ofDNA comprising using the nucleotide sequence of SEQ ID NO:
 1. 18. Apolynucleotide encoding a polypeptide having haloperoxidase activityobtained by the method of claim
 17. 19. A polypeptide havinghaloperoxidase activity encoded by the polynucleotide of claim 18.